Cationic Antimicrobial Peptides Bind Bacterial Acyl Carrier Proteins' Helix II, Proposing a Fatty Acid Synthesis Blockade

The escalating crisis of multidrug-resistant (MDR) pathogenic bacteria necessitates novel therapeutic strategies beyond conventional antibiotics. Cationic antimicrobial peptides (AMPs) are emerging as promising alternatives due to their broad-spectrum activity and diverse mechanisms. Bacterial acyl carrier proteins (ACPs), small (~9 kDa) highly acidic proteins, are crucial cofactors in bacterial fatty acid synthesis (FAS), a vital pathway for bacterial survival. Previous research hinted that human AMP LL-37 could target ACPs, affecting FAS and membrane integrity. This study further explores ACPs as a potential intracellular target for a broader range of AMPs and antibiofilm peptides (ABPs), aiming to identify common binding mechanisms.

Researchers investigated the interaction of various cationic antimicrobial and antibiofilm peptides with acyl carrier proteins (ACPs) from two significant pathogens: Pseudomonas aeruginosa ACP (PaACP) and Francisella novicida ACP (FnACP). They first characterized the folding of both ACPs using biophysical methods, noting differences in their requirements for divalent cations. Subsequently, they employed surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) spectroscopy to study the binding of several peptides, including LL-37, melittin, tritrpticin, indolicidin, puroindoline A, lactoferricin B, and IDR-1018, to both apo- and holo-forms of the ACPs. A control peptide, F5W-magainin 2, was also tested.

The study revealed distinct folding characteristics for the two ACPs: PaACP was found to be fully folded at neutral pH even without divalent cations, while FnACP remained unfolded at low ionic strength, requiring the addition of divalent cations like Ca2+ or Mg2+ to achieve a fully folded conformation. This difference was attributed to a unique His residue in FnACP involved in a stabilizing cation-π interaction. Biophysical SPR and NMR studies demonstrated that a broad range of cationic AMPs and ABPs, including LL-37, melittin, tritrpticin, indolicidin, puroindoline A, lactoferricin B, and IDR-1018, successfully bound to both apo- and holo-forms of the ACPs, whereas F5W-magainin 2 did not bind. Binding affinity was notably higher for holo-ACP (which contains the functional phosphopantothenate group) compared to apo-ACP. Furthermore, Arg-rich peptides exhibited a preference for binding over their Lys-rich analogs. NMR peak intensity perturbation data provided a critical insight: > Helix II of ACP, a region known to be directly involved in complex formation with bacterial FAS enzymes, consistently served as the common and primary recognition site for all binding peptides. This suggests a direct mechanism for interference with bacterial fatty acid synthesis.